The Hereditary Spastic Paraplegias (HSPs) are degenerative spinal cord disorders that cause disabling lower extremity spastic weakness. Recently, we discovered mutations in a novel gene (SPGSA/atlastin) as the cause of autosomal dominant SPG3A HSP. This advance has been translated into a clinically available diagnostic test for HSP and provides opportunities to expose HSP's molecular pathophysiology. Our proposal builds on this progress and focuses on 1) defining the SPG3A HSP phenotype and correlating clinical variatipn with SPG3A mutations and HSP modifying-gene polymorphisms; 2) understanding SPGSA/atlastin function by identifying its interacting factors; and 3) studying SPG3A pathogenesis in in vitro and animal models. Specific Aim 1 will characterize the phenotype and examine the basis of clinical variation in SPG3A HSP. The SPG3A phenotype can be quite variable and include childhood- and adult-onset uncomplicated HSP and HSP associated with lower motor neuron signs. Although clinical variation sometimes correlates with discrete SPGSA/atlastin mutation, variation between subjects with the same mutation indicates the action of modifying factors. There is precedent for benign HSP gene polymorphism to modify the HSP phenotype. Therefore, in addition to correlating SPG3A HSP phenotype variation with discrete SPG3A mutations, Specific Aim 1 will assess benign polymorphisms in SPGSA/atlastin, SPG4/spastin, and other HSP genes as "candidate modifying factors". Identifying disease modifying genes in HSP will expand our knowledge of HSP pathogenesis. Determining the SPGSA/atlastin genotype-phenotype correlations will suggest SPG3A functional domains and contribute to our studies of factors that interact with atlastin. Specific Aim 2 will provide insight into the function of SPGSA's encoded protein "atlastin" by identifying its interacting factors. Presently, clues to atlastin's function come from 3 sources: 1) its localization to cis-golgi membranes of cortical motor neurons; 2) it's homology to guanylate binding protein 1, a member of the dynamin family of GTPases that play essential roles in a wide variety of endosome trafficking events; and 3) its reported interaction in vitro with HPK/GCK-like kinase (HGK), a protein kinase in the c-Jun N-terminal kinase signaling (JNK) pathway. Specific Aim 2 will identify and analyze atlastin interacting proteins, investigate the reported atlastin-HGK interaction, and determine whether HSP-specific SPGSA/atlastin mutations change the proteins with which atlastin interacts. Specific Aim 3 will examine SPGSA/atlastin pathogenesis in vitro and in vivo. In view of atlastin's localization to cis-golgi, we are particularly interested in examining Golgi structure and function in in vitro and in vivo models. We will study morphology, differentiation, and atlastin intracellular location of cultured neurons bearing SPGSA/atlastin insufficiency (created through RNAi methods); and those with overexpression of wild-type and mutant SPG3A cDNA. We will study the behavior and neuropathology of SPGSA/atlastin mutation mice already created in our laboratory. Preliminary analysis of these animals reveals age-dependent hind limb motor impairment. By identifying SPG3A genotype-phenotype correlations, discovering SPGSA/atlastin-interacting factors, exposing metabolic cascades in which atlastin interacts, and examining in vitro and in vivo models of HSP, this investigation will provide insight into the causes and ultimately treatments for HSP and other motor neuron diseases including amyotrophic lateral sclerosis. [unreadable] [unreadable] [unreadable]